The long term objective of this research proposal is to gain new insights into the molecular and cellular events that are caused by encountering foreign antigens and generate the appropriate immune responses. The ligation of receptors on T cells with their counter receptors on APCs initiates cell-mediated immune responses. The molecular mechanisms by which multiple extracellularly engaged receptors regulated intracellular biochemical response is of prime importance in modern immunology. We have recently developed a highly sensitive and quantitative 3-dimensional (3- D) immunofluorescence workstation to study at the single cell level physiological immune interactions. Our previous studies at the single cell level showed that engaged receptors and their associated proteins cluster at the T-APC contact area. Early 3-D studies indicate that multiple adhesion and signaling molecules cluster ind distinct domains at the cell-cell contacts. In the present proposal we will use the new imaging system to study at the single cell level the detailed spatial and temporal molecular rearrangements that occur at the T-APC contacts during physiological interactions. We will then attempt to determine the mechanisms that are responsible for these regulated rearrangements and their functional significance for the relevant immune responses. The specific aims are: Aim 1. To study the temporal and 3-dimensional spatial redistribution of T cell receptors (TCR, CD4, FLA-1, CD28, CD45) and their colocalization with intracellular signaling and regulatory proteins that are implicated in regulating T cell responses (talin, PKCtheta, P-Tyr, grb2, lck, fyn, zap-70, PI-3K, PLCgamma1, and ras-GAP) in Ag-induced T-APC conjugates in order to determine their functional involvement in physiological immune interactions. Aim 2. To determine the role of CD28 and to identify its associated proteins during physiological Th-APC by repeating the experiments in Aim 1 with a cloned Th cell that does not express CD28 but retained expression of TCR, CD4, LFA-1 and CD45. To confirm causality by re- expressing either full length CD28 or a truncated CD28 that lacks its entire cytoplasmic domain. Aim 3. To determine by structure-function analysis the structural basis for the selective clustering of PKCtheta, but not any of the other expressed PKCs, at the cell contract along with CD28. To identify signals that cause this unique PKC translocation with the aid of well-defined PDGF-receptor signaling mutants. It is expected that these novel studies at the single cell level will significantly increase our understanding of the molecular events that occur early in the immune response and determine its outcome. This knowledge may be useful in the long term in enhancing immune surveillance and in improving the design of new vaccines.